cAMP is a nearly ubiquitous second messenger molecule that affects a multitude of cellular functions. In mammalian cells, two classes of adenylyl cyclase generate cAMP. Transmembrane adenylyl cyclases (tmACs) are tethered to the plasma membrane and regulated by heterotrimeric G proteins in response to hormonal stimuli (for review, see Hanoune et al., “Regulation and Role of Adenylyl Cyclase Isoforms,” Annu. Rev. Pharmacol. Toxicol. 41:145-174 (2001)). A second source of cAMP, the more recently described “soluble” adenylyl cyclase (sAC), resides in discrete compartments throughout the cell (Zippin et al., “Compartmentalization of Bicarbonate-Sensitive Adenylyl Cyclase in Distinct Signaling Microdomains,” FASEB J. 17:82-84 (2003)) and is regulated by the intracellular signaling molecules, bicarbonate (Chen et al., “Soluble Adenylyl Cyclase as an Evolutionarily Conserved Bicarbonate Sensor,” Science 289:625-628 (2000)) and calcium (Jaiswal et al., “Calcium Regulation of the Soluble Adenylyl Cyclase Expressed in Mammalian Spermatozoa,” Proc. Natl. Acad. Sci. USA 100:10676-10681 (2003); Litvin et al., “Kinetic Properties of ‘Soluble’ Adenylyl Cyclase. Synergism Between Calcium and Bicarbonate,” J. Biol. Chem. 278:15922-15926 (2003)).
cAMP elicits its cellular effects by activation of three known classes of effector proteins: exchange proteins activated by cAMP (EPAC), cyclic nucleotide gated ion channels, and protein kinase A (PKA). A subset of these targets resides at the plasma membrane, where they exist in macromolecular signaling complexes that also include a G protein coupled receptor, its transducing G protein, and the source of cAMP, a tmAC isoform (Davare et al., “A Beta2 Adrenergic Receptor Signaling Complex Assembled With the Ca2+ Channel Cav1.2,” Science 293:98-101 (2001)). The cAMP generated by tmACs appears to act locally (Rich et al., “Cyclic Nucleotide-Gated Channels Colocalize With Adenylyl Cyclase in Regions of Restricted cAMP Diffusion,” J. Gen. Physiol. 116:147-161 (2000); Rich et al., “A Uniform Extracellular Stimulus Triggers Distinct cAMP Signals in Different Compartments of a Simple Cell,” Proc. Natl. Acad. Sci. USA 98:13049-13054 (2001); Zaccolo et al., “Discrete Microdomains With High Concentration of cAMP in Stimulated Rat Neonatal Cardiac Myocytes,” Science 295:1711-1715 (2002)), most likely restricted by phosphodiesterase “firewalls” (Zaccolo et al., “Discrete Microdomains With High Concentration of cAMP in Stimulated Rat Neonatal Cardiac Myocytes,” Science 295:1711-1715 (2002); Mongillo et al., “Fluorescence resonance Energy Transfer-Based Analysis of cAMP Dynamics in Live Neonatal Rat Cardiac Myocytes Reveals Distinct Functions of Compartmentalized Phosphodiesterases,” Cir Res 95(1):65-75 (2004)), which define the limits of these cAMP signaling microdomains. However, targets of cAMP do not solely reside at the plasma membrane. EPAC is localized to the nuclear membrane and mitochondria (Qiao et al., “Cell Cycle-Dependent Subcellular Localization of Exchange Factor Directly Activated by cAMP,” J. Biol. Chem. 277:26581-26586 (2002)), and PKA is tethered throughout the cell by a class of proteins called AKAP (A-kinase-anchoring proteins; Michel et al., “AKAP Mediated Signal Transduction,” Annu. Rev. Pharmacol. Toxicol. 42:235-257 (2002)). The observation that cAMP does not diffuse far from tmACs (Bacskai et al., “Spatially Resolved Dynamics of Camp and Protein Kinase A Subunits in Aplysia Sensory Neurons,” Science 260:222-226 (1993); Zaccolo et al., “Discrete Microdomains With High Concentration of cAMP in Stimulated Rat Neonatal Cardiac Myocytes,” Science 295:1711-1715 (2002)) reveals that there must be another source of cAMP modulating the activity of these distally localized targets.
Soluble adenylyl cyclase (sAC; Buck et al., “Cytosolic Adenylyl Cyclase Defines a Unique Signaling Molecule in Mammals,” Proc. Natl. Acad. Sci. USA 96:79-84 (1999); U.S. Pat. No. 6,544,768 to Buck et al.; International Publication No. WO 01/85753) is widely expressed in mammalian cells (Sinclair et al., “Specific Expression of Soluble Adenylyl Cyclase in Male Germ Cells,” Mol. Reprod. Dev. 56:6-11 (2000)). Unlike tmACs, sAC is G protein insensitive (Buck et al., “Cytosolic Adenylyl Cyclase Defines a Unique Signaling Molecule in Mammals,” Proc. Natl. Acad. Sci. USA 96:79-84 (1999)), and among mammalian cyclases, it is uniquely responsive to intracellular levels of bicarbonate (Chen et al., “Soluble Adenylyl Cyclase as an Evolutionarily Conserved Bicarbonate Sensor,” Science 289:625-628 (2000)). The ubiquitous presence of carbonic anhydrases ensures that the intracellular bicarbonate concentration (and sAC activity) will reflect changes in pH (Pastor-Soler et al., “Bicarbonate-Regulated Adenylyl Cyclase (sAC) is a Sensor That Regulates pH-Dependent V-ATPase Recycling,” J. Biol. Chem. 278:49523-49529 (2003)) and/or CO2. Because CO2 is the end product of energy-producing metabolic processes, sAC is poised to function as a cell's intrinsic sensor of metabolic activity (Zippin et al., “CO(2)/HCO(3)(−)-Responsive Soluble Adenylyl Cyclase as a Putative Metabolic Sensor,” Trends Endocrinol. Metab. 12:366-370 (2001)). sAC possesses no transmembrane spanning domains (Buck et al., “Cytosolic Adenylyl Cyclase Defines a Unique Signaling Molecule in Mammals,” Proc. Natl. Acad. Sci. USA 96:79-84 (1999)) and is distributed to subcellular compartments containing cAMP targets (Zippin et al., “Compartmentalization of Bicarbonate-Sensitive Adenylyl Cyclase in Distinct Signaling Microdomains,” FASEB J. 17:82-84 (2003)) that are distant from the plasma membrane. sAC was also found localized inside the mammalian cell nucleus (Zippin et al., “Compartmentalization of Bicarbonate-Sensitive Adenylyl Cyclase in Distinct Signaling Microdomains,” FASEB J. 17:82-84 (2003)).
Although cAMP has been well known as a ubiquitous second messenger molecule affecting many different cellular functions, the source of cAMP in certain cellular processes and its connection to those processes have remained undefined.
The present invention is directed to overcoming these and other deficiencies in the art.